US11441114B2 - Liquid feed device, and cell culture device and method using same - Google Patents
Liquid feed device, and cell culture device and method using same Download PDFInfo
- Publication number
- US11441114B2 US11441114B2 US16/316,908 US201716316908A US11441114B2 US 11441114 B2 US11441114 B2 US 11441114B2 US 201716316908 A US201716316908 A US 201716316908A US 11441114 B2 US11441114 B2 US 11441114B2
- Authority
- US
- United States
- Prior art keywords
- liquid
- valve
- pump
- supply
- supply pipe
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active, expires
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M37/00—Means for sterilizing, maintaining sterile conditions or avoiding chemical or biological contamination
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M1/00—Apparatus for enzymology or microbiology
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/06—Nozzles; Sprayers; Spargers; Diffusers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/48—Automatic or computerized control
Definitions
- the present invention relates to a cell culture device and method to culture cells, and in particular relates to a liquid feed technique therefor.
- CPC Cell Processing Center
- devices to automate cell culture processes in closed systems have been developed. They achieve automation of cell culture processes and lowering of the risk of biological contamination by using closed system culture containers that do not require manipulation of opening and closing lids of the culture containers.
- There is a method for an automatic culture device in which dispensers are mechanized and liquid addition is performed in operations performed in an interlocking manner for preparative isolation and transfer similar to manual manipulation, but the device is increased in size because it is necessary to install the entire device in a sterile environment.
- there is a method of simultaneously performing quantitative determination and liquid feeding using a pump by connecting spaces from a liquid bottle to a culture plate using a disposable tube if the pump is used for dispensing operation.
- PTL 1 discloses an automatic culture device in which a mechanized dispenser is used
- PTL 2 discloses an automatic culture device in which pump liquid feed is used.
- a pump for dispensing, methods to cause liquids to flow in one direction are used, in which a bottle containing a liquid medium to be the source of liquid feed or a liquid medium in which cells or a biological sample are suspended (hereinafter, referred to as a cell suspension) is arranged upstream of the pump, and a container such as a culture plate is arranged downstream of the pump.
- a cell suspension passes through the inside of the pump, there is a concern in some cases that an excessive load occurs to the cells or biological sample after the passage due to a pressure change accompanying liquid feed.
- An object of the present invention is to solve such problems and to provide a liquid feed device that reduces the stress load on cells or biological samples contained in a liquid at the time of liquid feed, and a cell culture device and method using the same.
- the present invention provides a liquid feed device configured to include: a first liquid containing unit that contains a first liquid; a second liquid containing unit that contains a second liquid; a pump connected between the first liquid containing unit and the second liquid containing unit; a receptacle connected downstream of the second liquid containing unit; a first supply pipe that supplies the first liquid to the receptacle through the pump; and a second supply pipe that supplies the second liquid to the receptacle.
- the present invention provide a cell culture device configured to include: a thermostat; a culture container arranged in the thermostat; a liquid feed device that feeds and discharges a liquid to and from the culture container; and a control unit that controls the thermostat and the liquid feed device, wherein the liquid feed device has: a first liquid containing unit that contains a first liquid; a second liquid containing unit that contains a second liquid; a pump connected between the first liquid containing unit and the second liquid containing unit; a first supply pipe that supplies the first liquid to the culture container through the pump; and a second supply pipe that supplies the second liquid to the culture container.
- the present invention provides a cell culture method that performs cell culture using, as a liquid feed device that feeds and discharges a liquid to and from a culture container arranged in a thermostat, a liquid feed device with a configuration having: a first liquid containing unit that contains a first liquid; a second liquid containing unit that contains a second liquid; a pump connected between the first liquid containing unit and the second liquid containing unit; a first supply pipe that supplies the first liquid to the culture container through the pump; and a second supply pipe that supplies the second liquid to the culture container.
- the stress load on cells or biological samples contained in a liquid at the time of liquid feed can be reduced.
- FIG. 1 is a figure of one configuration of a liquid feed device in Example 1.
- FIG. 2 is a figure showing one example of a control time chart about the liquid feed device in Example 1.
- FIG. 3 is a figure of another configuration of the liquid feed device in Example 1.
- FIG. 4 is a figure showing another example of a control time chart about the liquid feed device in Example 1.
- FIG. 5 is a figure of one configuration of the liquid feed device and a cell culture device in Example 1.
- FIG. 6 is a figure showing one example of a control flow of an automatic culture device in Example 1.
- FIG. 7 is a figure showing a control time chart example of the automatic culture device in Example 1.
- FIG. 8 is a figure of one configuration of a liquid feed device in Example 2.
- FIG. 9 is a figure showing control of the liquid feed device in Example 2 and a weight measurement data example.
- FIG. 10 is a figure of one configuration of a liquid feed device in Example 3.
- FIG. 11 is a figure showing a control flowchart example about the liquid feed device in Example 3.
- FIG. 12 is a figure of another configuration of the liquid feed device in Example 3.
- liquid feed pipes to feed liquids and gas feed pipes to feed gases are collectively called supply pipes, and these supply pipes function as liquid feed pipes or gas feed pipes.
- Example 1 is an example of: a liquid feed device configured to include: a first liquid containing unit that contains a first liquid; a second liquid containing unit that contains a second liquid; a pump connected between the first liquid containing unit and the second liquid containing unit; a receptacle connected downstream of the second liquid containing unit; a first supply pipe that supplies the first liquid to the receptacle through the pump; and a second supply pipe that supplies the second liquid to the receptacle; and a cell culture device using the liquid feed device.
- the liquid feed device and the cell culture device using it according to Example 1 are explained with reference to FIG. 1 to FIG. 7 . They are explained in the following order: the configurations of the liquid feed device, a cell culture container and an automatic cell culture device; and manipulation of cell culture.
- FIG. 1 is a figure showing one configuration of the liquid feed device in the first example.
- a first liquid bottle 2 which is a first liquid containing unit to contain a liquid can keep its inner space airtightly with its lid (lid).
- a gas pressure adjusting pipeline 3 for gas pressure adjustment provided to the lid is open to the external air through a filter 4 provided to an open end thereof and having a mesh size of 0.22 ⁇ m.
- a supply pipe 5 provided to the lid has an open end inside the first liquid bottle 2 , which serves as a liquid discharge port in contact with the liquid in the liquid bottle 2 .
- One end of a pump 6 is connected to the other end of the supply pipe 5 , and the other end of the pump 6 is connected to a supply pipe 7 .
- the supply pipe 5 and supply pipe 7 are constituted by one rubber tube, for example, and its flow path is connected to a fluid drive unit of the pump 6 .
- a liquid feed destination of liquid feed of this supply pipe 7 is a liquid supply pipe 21 to supply a receptacle 8 with a liquid.
- the supply pipe 5 and supply pipe 7 are collectively called a first supply pipe in some cases.
- a branch point 9 is provided above the liquid surface of the liquid in the first liquid bottle contained in the first liquid bottle 2 of the supply pipe 5 . This is for, as explained below, causing a liquid in the supply pipe 5 between the position of the branch point 9 and the first liquid bottle 2 to return to the liquid bottle 2 by means of the potential energy generated due to a difference in elevation of the liquid when a first gas introducing valve 10 is opened and a gas is fed. This is because if a liquid is kept contained in the supply pipe 5 , clogging occurs when the liquid dries, and this should be prevented.
- the first gas introducing valve 10 opens and closes a pipe connected to the branch point 9 and a filter 11 .
- a valve mechanism used for this first gas introducing valve 10 is suitably a solenoid valve.
- a so-called solenoid valve has a mechanism in which a rubber tube is attached to and sandwiched by a part that opens and closes due to an effect of an electromagnet, and a pipe portion is opened and closed by elastically deforming the rubber tube according to turning on/off of the solenoid valve.
- elements referred to as valves means solenoid valves.
- the filter 11 is a filter having a mesh size of 0.22 ⁇ m and is in contact with the external air.
- a second liquid bottle 12 which is a second liquid containing unit to contain a liquid keeps its inner space airtightly with a lid.
- the second liquid bottle 12 is connected to a second gas introducing valve 15 via a filter 14 having a mesh size of 0.22 ⁇ m and provided to an open end of a gas pressure adjusting pipeline 13 which is a gas pipeline 13 provided to the lid.
- the gas pipeline 13 reaches a branch 16 through the second gas introducing valve 15 .
- the first gas introducing valve 10 and second gas introducing valve 15 constitute a gas introducing valve that introduces a gas into the second liquid bottle 12 which is the second liquid containing unit.
- a branch point 16 is provided at an intermediate position of the supply pipe 7 extending from one end of the pump 6 , and the abovementioned gas pipeline 13 branches off at the branch point 16 .
- the gas pipeline 13 is opened and closed by the second gas introducing valve 15
- the supply pipe 7 is opened and closed by a first supply valve 17 inserted between the branch point 16 and a branch point 20 .
- a supply pipe 18 provided to the lid of the second liquid bottle 12 has an open end inside the second liquid bottle 12 , which serves as a liquid discharge port in contact with a liquid.
- this supply pipe 18 may be called a second supply pipe.
- a second supply valve 19 is connected at an intermediate position of the supply pipe 18 , and the second supply valve 19 opens and closes the supply pipe 18 .
- the branch point 20 of the supply pipe 7 and the supply pipe 18 is connected to the liquid supply pipe 21 to the receptacle 8 closed off by a highly airtight lid.
- the receptacle 8 is provided with a filter 22 for gas pressure adjustment.
- An open portion of this liquid supply pipe 21 and the branch point 20 are provided above the liquid surface of the liquid in the second liquid bottle 12 .
- Opening and closure of the abovementioned pump 6 , first gas introducing valve 10 , second gas introducing valve 15 , first supply valve 17 and second supply valve 19 , that is, operation of opening and closure thereof is controlled by a controller 23 which is a control unit.
- the controller 23 can be realized by execution of a program by a central processing unit (CPU).
- the liquid feed device 1 performs feeding of a liquid in the first liquid bottle 2 in the following manner.
- the flow rate of the pump 6 is assumed to be approximately Q. If the first gas introducing valve 10 , second gas introducing valve 15 and second supply valve 19 are closed, and the first supply valve 17 is opened, and then the pump 6 is activated, the pump 6 feeds a gas in the supply pipe 5 , the liquid in the first liquid bottle 2 continuous with the gas passes the supply pipe 5 and liquid feed is thus started.
- the liquid passes the branch point 9 , and upon completion of supply of a predetermined liquid volume A from the first liquid bottle 2 , the pump 6 is stopped. When the pump 6 is stopped, the pipe is blocked due to the internal structure of the pump 6 , and the liquid does not move.
- a gas is introduced through the filter 11 , and additionally a liquid (return volume B) which is in the supply pipe 5 on the first liquid bottle 2 side relative to the position of the branch point 9 returns to the liquid bottle 2 due to the energy resulting from a difference in elevation.
- a liquid on the pump 6 side relative to the branch point 9 maintains its stopped state due to the abovementioned internal structure of the pump 6 .
- This liquid on the pump 6 side becomes a target predetermined liquid feed volume.
- a gas is sequentially introduced through the filter 11 , and additionally a liquid moves through the supply pipe 7 toward the liquid supply pipe 21 .
- the front end of the liquid arrives at the receptacle 8 , addition of the liquid is started thereby, and if the rear end of the liquid arrives at the receptacle 8 , the pump 6 is stopped.
- the liquid feed device 1 performs feeding of a liquid in the second liquid bottle 12 in the following manner. If the first supply valve 17 is closed, the first gas introducing valve 10 , second gas introducing valve 15 and second supply valve 19 are opened, and then the pump 6 is activated, a gas is introduced through the filter 11 , and additionally the pump 6 starts pressurization through the branch point 9 on the liquid in the second liquid bottle 12 . Due to pressure propagation through the gas phase, the liquid in the second liquid bottle 12 passes the second supply valve 19 through the supply pipe 18 , and liquid feed to the receptacle 8 is started thereby.
- the liquid passes the branch point 20 , upon completion of supply of a predetermined liquid volume C from the second liquid bottle 12 , the pump 6 is stopped, and additionally the second gas introducing valve 15 and second supply valve 19 are closed. Due to the action of the respective valves, the supply pipe 18 is blocked, and a liquid does not move therethrough. At this time, the pipe upstream of the branch point 20 in the supply pipe 18 is filled with a liquid, and the liquid volume equivalent to the inner volume of the pipe determined by the length and diameter of the pipe is assumed to be D.
- the pump 6 is activated, a gas is introduced through the filter 11 , and the gas moves through the branch point 16 and first supply valve 17 to move a liquid downstream of the position of the branch point 20 on the container side to the receptacle 8 .
- the front end of the liquid arrives at the receptacle 8 , addition of the liquid is started thereby, and if the rear end of the liquid arrives at the receptacle 8 , the pump 6 is stopped.
- FIG. 2 shows one example of a control time chart about the liquid feed device of the present example.
- the first supply valve 17 is opened at “START”, then the pump 6 is activated, and the liquid feed is started thereby.
- the first gas introducing valve 10 is opened.
- a length of time which is longer than that required for the rear end of the liquid to arrive at the receptacle 8 is set as the length of time during which the pump 6 is activated, and the pump 6 is activated for the length of time. After the given length of time, the pump 6 is stopped, and then all the valves are closed.
- the first gas introducing valve 10 , second gas introducing valve 15 and second supply valve 19 are opened, then the pump 6 is activated, and the liquid feed is started thereby.
- the second supply valve 19 and second gas introducing valve 15 are closed, and then the first supply valve 17 is opened.
- a length of time longer than that required for the rear end of the liquid to arrive at the receptacle 8 is set for the pump 6 , and the pump 6 is activated for the length of time.
- the pump 6 is stopped.
- the second gas introducing valve 15 is opened, and then all the valves are closed.
- the timing of opening and closure of each valve after completion of supply of the predetermined liquid volume C from the first liquid bottle 2 and after activation of the pump 6 is once stopped is important for feeding a liquid accurately.
- To first close the second supply valve 19 and second gas introducing valve 15 and stop the flows of a liquid and a gas keeps an increased pressure in the second liquid bottle 12 within the bottle 2 , and by opening the second gas introducing valve 15 after the rear end of the liquid arrives at the receptacle 8 , the gas phase pressure in the bottle is propagated to the container 8 side through the first supply valve 17 , and this results in the pressure to be applied onto the liquid in the pipe being maintained at the normal pressure.
- a liquid that is desired to avoid as much as possible influence of pressure changes in the liquid to be the source of liquid feed is contained in the second liquid bottle 12
- a liquid that is less susceptible to influence of pressure changes in the liquid to be the source of liquid feed is contained in the first liquid bottle 2 ; thereby, the liquid that is desired to avoid as much as possible influence of pressure changes can be fed to a target container with reduced influence of passing though the pump 6 ; on the other hand, the liquid that is not susceptible to influence of pressure changes can pass the pump 6 , and be fed to a target container quantitatively repetitively.
- the liquid that is desired to avoid as much as possible influence of pressure changes is contained in the second liquid bottle 12 arranged downstream of the pump 6 and is fed to the target container 8 by pressure propagation via a gas phase; thereby, it can be fed to the target container with reduced influence of passing the inside of the pump 6 .
- the liquid that is not susceptible to influence of pressure changes is contained in the first liquid bottle 2 arranged upstream of the pump 6 , and fed by passing the pump 6 ; as a result, it can be fed to the target container repetitively by quantitative control according to the flow rate accuracy of the pump 6 .
- the suspension of cells or the like can be fed to the receptacle without passing the pump, and the load on the cells or biological sample can be reduced.
- a liquid medium or the like is contained in the first liquid bottle 2 , the liquid medium can be fed quantitatively repetitively.
- the pump 6 is suitably a roller pump, but another form of pump such as a diaphragm pump or a gear pump can also be applied as the pump 6 .
- a roller pump which is a so-called peristaltic pump or tube pump has a mechanism in which a rubber tube is wound around a roller attached to a motor rotational shaft and rotation of the motor elastically deforms the rubber tube to feed a gas or liquid therein. It is necessary to ensure sterility of a tube for liquid feeding in a cell culture device, and a roller pump which allows replacement of a tube at the time of use is useful. If internal sterilization is possible before use, any type of liquid feed pump can be used.
- a configuration to not move a liquid inside is necessary at the time of stopping the pump, but by configuring a pipeline via a check valve that limits a flow toward a liquid feed bottle side before or after the pump at the time of use of the pump during which a liquid moves, it can be applied to the device of the present example.
- liquid feed device of the present example in repetitively feeding a liquid in the second liquid bottle 12 , the liquid surface of the liquid at the first time is inside the second liquid bottle 12 , but the liquid surface at the time of next liquid feed is at the position of the branch point 20 in the supply pipe 18 .
- the liquid volume is, as mentioned above, the volume D equivalent to the inner volume of the pipe upstream of the branch point 20 in the supply pipe 18 , but at the time of first liquid feed, liquid feed control may be performed taking into consideration the difference volume.
- liquid feed device of the present example it is also possible to perform liquid feed after stirring a liquid to be fed by another liquid feed method.
- the configuration of a liquid feed device in FIG. 3 is the same as the configuration in the example shown in FIG. 1 , and configuration parts related to the second liquid bottle 12 are illustrated in detail.
- a liquid By repetitively performing these pressurization and suction operations, a liquid can be stirred highly efficiently. This is pronounced if a liquid feed target is a liquid in which a liquid composition is not uniform, and for example in a case of a cell suspension in which cells are suspended in a medium, a manual stirring operation is performed by generating a convection current in the liquid by pushing and pulling a piston of a dispenser before cell seeding.
- a manual stirring operation is performed by generating a convection current in the liquid by pushing and pulling a piston of a dispenser before cell seeding.
- liquid feed control can be realized by temporal control based on a flow rate of the pump and a target liquid volume such that a liquid with the target liquid volume passes a space equivalent to the pipe volume estimated from the length and diameter of a pipe to a target container.
- FIG. 4 shows a control flowchart of a method of feeding a liquid after being stirred while ensuring reproducibility of liquid feeding in Example 1.
- the first supply valve 17 is opened at “START”, then the pump 6 is activated, and the feeding of the first liquid is started thereby.
- the first gas introducing valve 10 is opened.
- a length of time which is longer than that required for the rear end of the liquid to arrive at the receptacle 8 is set as the length of time during which the pump 6 is activated, and the pump 6 is operated for the length of time. After the given length of time, the pump 6 is stopped, and then all the valves are closed.
- the first gas introducing valve 10 , second gas introducing valve 15 and second supply valve 19 are opened, and then the pump 6 is activated.
- the pump 6 is activated under a suction condition opposite to the normal suction condition, the inside of the second liquid bottle 12 is brought to a negative pressure, and a liquid in the second liquid bottle 12 is stirred. Thereafter, the pump 6 is activated under the normal condition, and liquid feed is started thereby.
- the supply valve 19 and second gas introducing valve 15 are closed, and then the first supply valve 17 is opened.
- a length of time longer than that required for the rear end of the liquid to arrive at the receptacle 8 is set for the pump 6 , and the pump 6 is activated for the length of time. After the given length of the time, the pump 6 is stopped, and then all the valves are closed. Here, similar to FIG. 2 , after once second gas introducing valve 15 is opened, all the valves may be closed.
- the criterion of the amount of speed change from a container having a larger diameter to a pipe having a small diameter is desirably 10,000% or smaller. That is, the ratio of the diameter D 1 of a liquid bottle to the diameter D 2 of a supply pipe is recommended to be 1,000% or smaller.
- the shape of a liquid bottle is generally cylindrical. It is normally manufactured to have an inner diameter which is smaller at the bottom surface than at the opening, and in addition, the bottom surface portion more preferably has a triangular pyramid shape.
- a triangular pyramid shape that is, a cone-like shape so as to arrange the supply pipe closer to the bottom portion, the remaining volume at the time of discharge from the liquid bottle to the supply pipe can be reduced.
- FIG. 5 is a figure showing one configuration example of an automatic cell culture device 31 using the liquid feed device 1 of Example 1.
- the cell culture device of the present example has a configuration having: a thermostat; a culture container arranged in the thermostat; a liquid feed device that feeds and discharges a liquid to and from the culture container; and a control unit that controls the thermostat and the liquid feed device, wherein the liquid feed device has: a first liquid containing unit that contains a first liquid; a second liquid containing unit that contains a second liquid; a pump connected between the first liquid containing unit and the second liquid containing unit; a first supply pipe that supplies the first liquid to the culture container through the pump; a second supply pipe that supplies the second liquid to the culture container; and a gas introducing valve that introduces a gas into the second liquid containing unit, and the present example relates to a cell culture method using the cell culture device.
- a thermostat 32 contains cell culture containers such as a first culture container 80 or a second culture container at a culture temperature optimal for cell culture.
- a refrigerator 33 contains elements such as a replenishment bottle 69 that are required to be kept at a low temperature.
- a first medium bottle 34 which is a first liquid containing unit to contain a seeding medium can keep its inner space airtightly with its lid.
- the first medium bottle 34 is provided with a gas pressure adjusting pipe 35 for gas pressure adjustment that is provided to one of lids.
- a filter 36 with a mesh size of 0.22 ⁇ m provided to an open end is installed and open to a gas phase of the thermostat 32 .
- One end of a supply pipe 37 that is provided to the lid and is to function as a first supply pipe has an open end inside the first medium bottle 34 , contacts the seeding medium and serves as a liquid discharge port.
- a first control valve 38 controls a flow in the supply pipe 37 .
- the supply pipe 37 is connected to a common pipe 42 mentioned below via a branch point 39 .
- the branch point 39 is provided above the liquid surface of a liquid contained in the first medium bottle 34 .
- the configuration of a second medium bottle 43 that contains a replacement medium is similar to the first medium bottle.
- One end of a supply pipe 44 has an open end inside the second medium bottle 43 , contacts the replacement medium, and serves as a liquid discharge port.
- a second control valve 45 controls a flow in the supply pipe 44 .
- the supply pipe 44 is connected to the common pipe 42 via a branch point 46 .
- the upstream of the common pipe 42 is connected to a first gas pressure adjusting valve 76 , and the first gas pressure adjusting valve 76 is connected to a gas common pipe 47 mentioned below.
- a pump 41 is connected to the downstream of the common pipe 42 , and also a first gas introducing valve 40 is connected thereto.
- a discharge portion of a humidifying bottle 48 is connected to the first gas introducing valve 40 , and an introducing portion of the humidifying bottle 48 branches to be connected to the gas common pipe 47 via a second gas pressure adjusting valve 49 .
- Another branch is connected to a pressure control valve 51 via a filter 50 , and to the upstream thereof, a mixed gas cylinder 52 containing CO 2 and O 2 is connected.
- the gas cylinder 52 is a cylinder containing CO 2 gas filling it while being pressurized at a gas concentration optimized for cell culture, is aimed for pH value adjustment of a liquid medium in the cell culture, and allows gas replacement through a surface of the liquid medium with CO 2 gas.
- the humidifying bottle 48 contains sterile water, and feeds CO 2 gas humidified by being caused to pass through the sterile water to a culture container; thereby, it is possible to prevent condensation of liquid medium components due to evaporation of the liquid medium. Thereby, CO 2 gas derived from the cylinder 52 is humidified to an optimal humidity and is kept waiting in the humidifying bottle 48 .
- a first connection 53 and a second connection 54 are provided at the liquid feed pump 41 .
- the first connection 53 and second connection 54 are connected to a second gas introducing valve 55 .
- the second gas introducing valve 55 plays a role of a bypass for the liquid feed pump 41 .
- the second connection 54 at the liquid feed pump 41 branches at a branch point 56 , and is connected to a third gas introducing valve 58 that performs open/close control of a gas feed pipe 57 and a gas feed valve 60 that performs open/close control of a gas feed pipe 59 .
- a cell suspension is contained in a cell bottle 61 which is a second liquid containing unit.
- the cell bottle 61 is connected to the gas feed pipe 59 via a filter 62 connected to a lid of the cell bottle 61 .
- Another pipe is a supply pipe 63 to function as a second supply pipe, having one end which is an open end inside the cell bottle 61 , contacts the cell suspension, and serves as a liquid discharge port.
- a third control valve 64 performs open/close control of the supply pipe 63 .
- the supply pipe 57 branches off from the supply pipe 63 at a branch point 65 , and is, at one end thereof, connected to a fourth control valve 68 that performs open/close control of a supply pipe 67 at a branch point 66 .
- a replenishment medium used for a replacement medium is contained in the replenishment bottle 69 .
- the replenishment bottle 69 is connected to a gas pressure adjusting pipe 71 via a filter 70 connected to a lid of the replenishment bottle 69 , and one end of the supply pipe 67 has an open end inside the replenishment bottle 69 , contacts the replenishment medium, and serves as a liquid discharge port. That is, pipes are configured such that the replacement medium in the replenishment bottle 69 is fed to the second medium bottle 43 by an action of the liquid feed pump 41 .
- the common pipe 42 is connected to the gas pressure adjusting pipe 71 of the replenishment bottle 69 and a gas pressure adjusting pipe 72 at the second medium bottle 43 via a third gas pressure adjusting valve 77 and the first gas pressure adjusting valve 76 . Furthermore, the common pipe 42 is connected to a gas bag 74 via a filter 73 and is connected to the abovementioned second gas pressure adjusting valve 49 .
- This gas bag 74 is provided with a check valve 75 and communicates with a gas phase in the incubator 32 .
- a gas in the gas cylinder 52 can be contained in the gas bag 74 by opening only the second gas pressure adjusting valve 49 .
- the gas pressure in the gas bag 74 is always maintained at atmospheric pressure. That is, it is configured such that the gas contained in the gas bag 74 contacts a gas phase in the replacement medium bottle 43 and a gas phase in the replenishment bottle 69 via the common pipe 42 . Furthermore, it is configured such that if the gas pressure adjusting valve 76 , first control valve 38 and second control valve 45 are opened, it contacts a liquid phase in the second medium bottle 43 and a liquid phase in the seeding medium bottle 34 .
- the supply pipe 57 is connected to a multibranched portion 78 that leads to a culture container, and is connected to a container open/close valve 82 for liquid feed in the first culture container 80 and a container open/close valve 83 for a second culture container 81 . Because both the first culture container 80 and the second culture container 81 have the same configuration, the first culture container 80 is representatively explained below about its configuration.
- the first culture container 80 is an airtight container having an external appearance that includes a body portion 84 and a lid portion 85 , and has an internal appearance that can contain a container 87 capable of containing and culturing a cell suspension 86 at the inner bottom portion of the body portion 84 .
- the lid portion 85 is provided with three penetrating ports. One of them is a liquid feed port 88 for adding a liquid to the container 87 and is connected to the abovementioned container open/close valve 82 . Another one of them is a discharge port 89 which contacts portions near the bottom surface of the container 87 and is for discharging a liquid, and the last one of them is a gas pressure adjusting port 90 .
- the liquid feed port 88 doubles as a gas feed port at the time of gas introduction, and because of this, open ends of the liquid feed port 88 and gas pressure adjusting port 90 are provided at the height at which they do not contact the liquid even if the container 87 is filled with the liquid.
- the gas pressure adjusting port 90 is connected to a fourth gas pressure adjusting valve 91 , and is connected to a trap bottle 93 through a multibranched portion 92 at which it branches off to the second culture container 81 .
- the trap bottle 93 is installed in the refrigerator 33 , and a gas phase that passes the trap bottle 93 is released to the refrigerator 33 via a filter 94 .
- a configuration of discharging, from the first culture container 80 or second culture container 81 , a liquid contained therein is explained.
- a liquid discharge pipe 98 is connected to a liquid discharge bottle 97 airtightly.
- the liquid discharge pipe 98 is connected to the exhaust port of a liquid discharge pump 96 via a discharge valve 99 .
- a first container discharge valve 101 for the first culture container 80 and a first container discharge valve 102 for the second culture container 81 that branch off at a multibranched portion 100 are connected to the suction port of the liquid discharge pump 96 .
- the first container discharge valve 101 is connected to the discharge port 89 at the first culture container 80 .
- the liquid discharge bottle 97 has a configuration of pipes in which due to an action of the liquid discharge pump 96 , a liquid is discharged from the container 87 at the first culture container 80 or the second culture container 81 .
- the various solenoid valves, pump 96 , thermostat 32 , refrigerator 33 or the like shown above are controlled by a controller 103 which is a control unit.
- FIG. 6 is a figure showing a flowchart of overall manipulation of cell culture in the cell culture device 31 controlled by the controller 103 which is a control unit shown in FIG. 5 .
- a flow path is installed in the thermostat 32 (S 01 ), and then the cell bottle 61 containing a separately prepared cell suspension, the medium bottle 34 containing a seeding medium and the replacement medium bottle 69 containing a replacement medium are connected to the flow path (S 02 ).
- the gas bag 74 is filled with a gas by automatic control (S 03 ).
- the cell suspension is fed from the cell bottle 61 (S 04 ).
- a seeding medium is fed from the seeding medium bottle 34 to the culture containers (S 05 ).
- a humidified gas is fed to the culture containers, and cells are left to stand still while being kept at a constant temperature (S 06 ).
- S 07 it is judged whether to start replacement of the liquid medium.
- the second medium bottle 43 is filled with a predetermined volume from the replenishment medium bottle 69 (S 08 )
- an old medium in the culture containers is discharged (S 09 ), and then a new liquid medium is fed from the second medium bottle 43 (S 10 ).
- FIG. 7 shows one example of a time chart of liquid feed/gas feed in the culture container 80 , which is controlled by the controller 103 in FIG. 5 .
- the horizontal axis indicates manipulation items and the time axis.
- operation timing of 18 solenoid valves, the first control valve 38 to container discharge valve 102 clearly shown in FIG. 5 , and roller pumps, the liquid feed pump 48 and liquid discharge pump 96 , and the like is shown.
- all the solenoid valves are turned off, and accordingly are closed, and all the pump are turned off. So liquid feed is stopped.
- the third control valve 64 is closed, the gas feed valve 60 is closed, and next the third gas introducing valve 58 is opened to start liquid feed of the liquid feed pump 41 , a cell fluid suspension is fed from the first port 88 of the culture container 80 through the container open/close valve 82 .
- the third port 90 communicates with external air through the trap bottle 93 , so the pressure inside the cell culture container 80 is adjusted to be the normal pressure.
- an effective method of bringing the internal pressure of the cell bottle 61 to the normal pressure may also be a method in which when the volume of a liquid that has arrived at the branch point 65 reached a target volume, and liquid feed of the liquid feed pump 41 is stopped, the third control valve 64 is closed and the second gas introducing valve 55 is opened, thereby introducing a gas in the gas bag.
- a volume of a cell suspension that is large enough to be able to be distributed to the plurality of cell culture containers is preliminarily contained in the cell bottle 61 . If in the abovementioned manipulation, the container open/close valve 82 is closed, the container open/close valve 83 in FIG. 5 is opened, the fourth gas pressure adjusting valve 91 is opened as appropriate, and the abovementioned operation is performed, the same volume of a cell fluid suspension can be fed to the cell culture container 81 .
- liquid feed of the liquid feed pump 41 is stopped.
- the first gas pressure adjusting valve 76 and first control valve are opened, the seeding medium in the supply pipe 37 is divided at the branch point 39 , and liquid on the seeding medium bottle 34 side returns to the bottle due to a difference in elevation.
- liquid feed of the liquid feed pump 41 is started, a seeding medium is fed from the first port 88 of the culture container 80 through the container open/close valve 82 .
- the third port 90 communicates with external air through the trap bottle 93 , so the pressure inside the cell culture container 80 is adjusted to be the normal pressure.
- a volume of a seeding medium that is large enough to be able to be distributed to the plurality of cell culture containers is preliminarily contained in the seeding medium bottle 34 . If in the abovementioned manipulation, the container open/close valve 82 is closed, the container open/close valve 83 in FIG. 5 is opened, the fourth gas pressure adjusting valve 91 is opened as appropriate, and the abovementioned operation is performed, the same volume of a seeding medium can be fed to the cell culture container 81 .
- the third port 90 to the filter 94 communicating with external air are opened, the pressure inside the culture container becomes a pressure adjusted to the pressure of the external air.
- the first gas introducing valve 40 is closed, next, the second gas introducing valve 55 is closed, and when the pressure in the culture container became comparable to the atmospheric pressure, the container open/close valve 82 and fourth gas pressure adjusting valve 91 are closed.
- the container open/close valve 83 and fourth gas pressure adjusting valve 91 are opened, and the abovementioned operation is performed, the culture container 81 is filled with CO 2 gas.
- the third gas pressure adjusting valve 77 , second control valve 45 , third gas introducing valve 58 and fourth control valve 68 are opened.
- the second medium bottle 43 becomes communicating with the replenishment medium bottle 69 via the liquid feed pump 41 .
- a liquid is fed from the liquid feed pump 41 in the direction opposite to the normal direction.
- liquid feed of the pump 41 is stopped.
- the target liquid volume is the volume of a liquid having been fed to the second medium bottle 43 and the volume of a liquid contained in the branch point 46 in the supply pipe 44 .
- the feeding volume of a pump is adjusted such that a volume of a liquid medium that is large enough to be distributed to the plurality of cell culture containers is preliminarily contained in the second medium bottle 43 .
- the volume of liquid medium that enables feeding of the liquid medium that is obtained by multiplying the liquid medium volume necessary when there is a plurality of cell culture containers by the number of times of replacement of the medium, consecutive replacement of the medium multiple times for the plurality of cell culture containers is possible.
- the container open/close valve 82 is closed, the container open/close valve 83 in FIG. 5 is opened, the container discharge valve 101 is closed, the container discharge valve 102 is opened, and the abovementioned operation is performed, a liquid medium can be discharged from the container in the culture container 81 .
- liquid feed of the liquid feed pump 41 is stopped.
- the first gas pressure adjusting valve 76 is opened, the replacement medium in the supply pipe 44 is divided at the branch point 46 , and liquid on the second medium bottle 43 side returns to the bottle due to a difference in elevation.
- liquid feed of the liquid feed pump 41 is started, a replacement medium is fed from the first port 88 of the culture container 80 through the container open/close valve 82 .
- the third port 90 communicates with external air through the trap bottle 93 , so the pressure inside the cell culture container 80 is adjusted to be the normal pressure.
- injection of a predetermined volume of the replacement medium is completed, the liquid feed pump 41 is stopped, and respective opened valves are turned off and closed to end the liquid feed.
- a volume of a liquid medium that is large enough to be able to be distributed to the plurality of cell culture containers is preliminarily contained in the second medium bottle 43 . If in the abovementioned manipulation, the container open/close valve 82 is closed, the container open/close valve 83 in FIG. 5 is opened, the fourth gas pressure adjusting valve 91 is opened as appropriate, and the abovementioned operation is performed, the same volume of a replacement medium can be fed to the cell culture container 81 .
- the culture container 80 is kept at an optimal culture temperature by the incubator 32 ; a cell suspension can be fed to a culture container by a liquid feed device; then, due to a gas feed mechanism, a liquid medium is maintained in a suitable CO 2 gas environment and under a suitable humidity condition; because cells grow adhering to the inner bottom surface of the container 87 , a liquid medium having been subjected to composition change along with culture can be suctionally discharged, and the cells and the liquid medium can be separated thereby; the second medium bottle 43 contained in the thermostat 32 in a suitable volume can be filled with a replacement medium and it can be contained therein while being preheated; next, a replacement medium is sequentially added to a culture container and medium replacement can be performed thereby.
- a liquid that is desired to avoid as much as possible influence of pressure changes in the liquid to be the source of liquid feed is contained in the second liquid bottle, and a liquid that is less susceptible to influence of pressure changes in the liquid to be the source of liquid feed is contained in the first liquid bottle; thereby, the liquid that is desired to avoid as much as possible influence of pressure changes can be fed to a target container with reduced influence of passing though the pump; on the other hand, the liquid that is not susceptible to influence of pressure changes can pass the pump, and be fed to a target container quantitatively repetitively.
- the liquid that is not susceptible to influence of pressure changes is contained in the first liquid bottle arranged upstream of the pump, and fed by passing the pump; as a result, it can be fed to the target container repetitively by quantitative control according to the flow rate accuracy of the pump.
- the liquid medium or the like is contained in the second liquid bottle, the liquid medium can be fed quantitatively repetitively.
- the liquid feed device of the present example and the cell culture device using the same provide the following effects.
- a cell suspension is fed first, and next a seeding medium is fed later.
- the cell suspension is fed first, there might be the cell suspension remaining as a small number of droplets in the liquid supply pipe communicating through to the culture container.
- the seeding medium or the like is fed thereafter, it passes through the liquid supply pipe, it can mix with the small number of droplets containing the cell suspension to arrive at a target container. Due to an effect of so-called prewashing, there is an effect of being able to reduce influence of cells remaining in the pipe.
- Example 2 in addition to having the configuration of the liquid feed device explained in Example 1, the liquid feed device can check a liquid feed volume by the following method. That is, in addition to having the configuration of Example 1, the liquid feed device of the present example includes weight sensors that respectively detect weights of a first liquid in a first liquid containing unit and a second liquid of a second liquid containing unit, and the control unit is configured to control the pump according to outputs of the weight sensors.
- FIG. 8 is a figure showing the configuration of a liquid feed device 110 in Example 2. Although it has a basic configuration which is the same as that in Example 1, a configuration of sensing values about weight changes of the liquid bottles is added to be capable of a method of checking liquid feed volumes. That is, it has a configuration which is the same as that Example 1 shown in FIG.
- the branch point 9 is provided above the liquid surface of the liquid in the first liquid bottle contained in the first liquid bottle 2 of the supply pipe 5 .
- a liquid in the supply pipe 5 between the position of the branch point 9 and the first liquid bottle 2 to return to the liquid bottle 2 by means of the potential energy generated due to a difference in elevation of the liquid when the first gas introducing valve 10 is opened and a gas is fed, and clogging can be prevented.
- the weight of a liquid bottle before and after liquid feed explained below it is desirable to cause the liquid to return to the liquid bottle 2 , and the volume of the liquid remaining in the pipe is 0.
- the configuration of the present example includes: a first weight sensor 111 that measures a weight of a set of the first liquid bottle 2 containing a liquid, the gas pressure adjusting pipeline 3 and the filter 4 ; and a fixing jig 112 that fixes the supply pipe 5 .
- a pipe material such as a highly flexible rubber tube is preferably used for the supply pipe 5 because constituent parts connected downstream of the branch point 9 do not impair weight measurement of the first liquid bottle 2 .
- the configuration of the present example includes: a second weight sensor 113 that measures a weight of a set of the second liquid bottle 12 containing a liquid, the gas pipeline 13 , the filter 14 and the supply pipe 18 ; a fixing jig 114 that fixes the gas pipeline 13 ; and a fixing jig 115 that fixes the supply pipe 18 .
- the fixing jigs 114 , 115 are both added for a purpose similar to that for the fixing jig 112 .
- the liquid feed device 110 of the present example feeds liquid and checks a liquid feed volume in the following manner.
- FIG. 9 the order of control in the present example is shown along the horizontal axis, and the vertical axis shows weight measurement values obtained by the weight sensors corresponding to it.
- weight measurement the liquid feed method performed for a liquid in the second liquid bottle 12 and changes in measurement values of the weight sensor 113 are explained.
- the weight measurement value of the second weight sensor 113 before liquid feed is measured, and this is assumed to be zero here.
- the first supply valve 17 is closed, the first gas introducing valve 10 , second gas introducing valve 15 and second supply valve 19 are opened, and then the pump 6 is activated, a gas is introduced through the filter 11 , and additionally the pump 6 starts pressurization through the branch point 9 on a liquid in the second liquid bottle 12 .
- the liquid in the second liquid bottle 12 passes the second supply valve 19 through the supply pipe 18 , liquid feed to the container 8 is started thereby, and along with it, the weight decreases.
- the pump 6 is stopped, the second supply valve 19 is closed, and next the second gas introducing valve 15 is closed.
- the supply pipe 18 is blocked, and a liquid does not move therethrough.
- the first supply valve 17 is opened and the pump 6 is activated, a gas is introduced through the filter 11 , and the gas moves through the branch point 16 and first supply valve 17 , a liquid downstream of the position of the branch point 20 on the container side starts moving and the volume of this liquid on the container 8 side is the object liquid feed volume.
- the front end of the liquid arrives at the receptacle 8 , addition of the liquid is started thereby, and if the rear end of the liquid arrives at the receptacle 8 , the pump 6 is stopped.
- the second gas introducing valve 15 and second supply valve 19 are opened, the liquid in the supply pipe 18 returns into the second liquid bottle 12 due to a difference in elevation, and the weight measurement value of the weight sensor 113 at that time is measured and liquid feed is ended.
- the target value weight E is a weight obtained by adding, to the weight-converted value of a liquid feed volume of a target liquid which is obtained based on its liquid volume and density, a weight F of a liquid filling the volume of the supply pipe 18 having a length from the branch point 20 to the liquid surface of a liquid in the liquid bottle 2 , the weight F being obtained based also on the density of the liquid. That is, a liquid feed volume of a liquid fed to the container 8 can be detected based on a weight measurement value obtained according to E-F.
- a liquid is fed repetitively, a successively decreasing weight can be handled as a fed liquid weight by executing the abovementioned manipulation.
- the liquid feed device of the present example can be used for checking a fed volume of a cell suspension at the time of cell seeding.
- execution of such dispensing steps is guaranteed by proficiency of workers and work implementation records.
- it is possible to guarantee sure execution results of cell culture steps by recording that change amounts of the weight of a cell bottle to be the source of liquid feed has reduced by a target weight and handling such a record as a work record of single event of dispensing.
- the liquid feed method controls liquid feed based on change amounts of the weight of a liquid to be fed as a result, and is thus can be said to provide good reproducibility. Additionally, if operation information obtained from the automatic device such as records of time during which all the pumps are actuated, records about voltages applied to the pumps or actuation time records of liquid surface sensors is organized, execution results of liquid feed and discharge can be guaranteed highly reliably.
- the liquid feed device of the present example to the cell culture device shown in Example 1, the load applied onto cells, a biological sample or the like due to pressure changes accompanying liquid feed can be reduced, furthermore liquid feed can be performed while at the time the liquid feed volume is controlled accurately, and furthermore automatic cell culture can be enabled.
- Example 3 in addition to having the configuration of the liquid feed device explained in Example 1, the liquid feed device can feed a very small liquid feed volume with good reproducibility by the following method. That is, this is an example of: a liquid feed device including a first liquid bottle containing a first liquid, a supply pipe through which the first liquid passes, a gas introducing valve, the gas introducing valve, the liquid bottle, a second liquid bottle containing a liquid that is desired to avoid as much possible influence of pressure changes, a supply pipe through which a second liquid passes, a pump and a target container, the liquid feed device being characterized by connecting the container at the downstream of the second liquid bottle, operating the pump to suction toward itself, and then feeding a liquid to target container; and furthermore a cell culture device including this liquid feed device.
- a liquid feed device including a first liquid bottle containing a first liquid, a supply pipe through which the first liquid passes, a gas introducing valve, the gas introducing valve, the liquid bottle, a second liquid bottle containing a liquid that is desired to avoid
- FIG. 10 is a figure showing one configuration of a liquid feed device 120 in Example 3, and shows a configuration a liquid that is desired to avoid as much as possible influence of pressure changes in a liquid to be the source of liquid feed is retained in a supply pipe in a required volume, and then the liquid is fed to a target container repetitively quantitatively.
- Example 1 it has the same configuration as that Example 1 in that it includes the first liquid bottle 2 , gas pressure adjusting pipeline 3 , filter 4 , supply pipe 5 , pump 6 , supply pipe 7 , receptacle 8 , branch point 9 , first gas introducing valve 10 , filter 11 , second liquid bottle 12 , filter 14 , supply pipe 18 , second supply valve 19 , branch point 20 , liquid supply pipe 21 , filter 22 and controller 23 .
- 121 includes a retention pipe 121 joined with the supply pipe 7 and the supply pipe 18 at the branch point 20 , and furthermore a first supply valve 122 that controls the liquid supply pipe 21 connected to the retention pipe 121 .
- the retention pipe 121 has a known length and pipe diameter, and may have the same diameter as that of other pipes which are the supply pipe 7 , supply pipe 18 and liquid supply pipe 21 .
- the liquid feed device 120 of the present example feeds liquid and checks liquid feed volume as shown next.
- FIG. 11 shows one example of a control flowchart of the present example.
- the first gas introducing valve 10 , and second supply valve 19 are opened, then the pump 6 is activated in the suction direction opposite to the target liquid feed direction, and liquid feed of the second liquid is started thereby.
- the pump 6 is activated in the suction direction opposite to the target liquid feed direction, and liquid feed of the second liquid is started thereby.
- activation of the pump 6 is promptly stopped.
- the first supply valve 122 is opened.
- a length of time which is longer than that required for the rear end of the liquid to arrive at the receptacle 8 is set as the length of time during which the pump 6 is activated, and the pump 6 is operated for the length of time.
- the pump 6 is stopped, and then all the valves are closed.
- the time of first liquid addition that is, at the time of feeding of a first liquid
- the first supply valve 122 is opened, then the pump 6 is activated in the target liquid feed direction, and the liquid feed of the first liquid contained in the first bottle 2 is started thereby.
- liquid feed device of the present example changes in the pressure applied to a cell suspension at the time of cell seeding can be made small, and furthermore the device configuration can also be simplified.
- a retention pipe is used and a liquid such as a cell suspension that is desired to avoid as much as possible influence of pressure changes and a liquid such as a liquid medium which is free of the influence of pressure changes share a supply pipe; thereby, an effect of being able to reduce control valves for controlling the supply pipe is provided.
- FIG. 12 is a figure showing a variant configuration example in which the basic configuration is the same as the liquid feed device 120 shown in FIG. 10 , but check valves 131 , 132 are provided along the illustrated direction in place of the second supply valve 19 and first supply valve 122 .
- the check valves 131 , 132 due to the check valves 131 , 132 , the direction of liquid feed of the supply pipe 18 is always in the suction direction relative to the liquid feed pump 6 , and a flow never occurs in the pressurizing direction.
- the direction of liquid feed of the liquid supply pipe 21 is always in the pressurizing direction relative to the liquid feed pump 6 , and a flow never occurs in the suction direction.
- the retention pipe 121 is changed as appropriate according to the target liquid feed volume, and if the target liquid feed volume is larger, the pipe diameter should be increased to avoid an excessive pipe length. On the other hand, if the target liquid feed volume is very small and accuracy is required, it is also useful to reduce the pipe diameter and reduce the internal surface area of the retention pipe 121 .
- a liquid that is desired to avoid as much as possible influence of pressure changes in the liquid to be the source of liquid feed is contained in the second liquid bottle, and a liquid that is less susceptible to influence of pressure changes in the liquid to be the source of liquid feed is contained in the first liquid bottle; thereby, the liquid that is desired to avoid as much as possible influence of pressure changes can be fed to a target container with reduced influence of passing though the pump; on the other hand, the liquid that is not susceptible to influence of pressure changes can pass the pump, and be fed to a target container quantitatively repetitively.
- the present invention is not limited to the abovementioned examples, but incorporates various variants.
- the abovementioned examples are explained in detail for better understanding of the present invention, but are not necessarily limited to those including all the explained configurations.
- part of the configuration of an example can be replaced with the configuration of another example, and also the configuration of an example can be added to the configuration of another example.
- part of the configuration of each example can be added to, deleted from and replaced by another configuration.
- functions controller which is a control unit
- functions controller which is a control unit
- they may be realized by hardware by designing them partially or entirely for example in an integrated circuit, and so on. That is, all the functions or some of the functions of processing units may be realized for example by an integrated circuit such as an ASIC (Application Specific Integrated Circuit) or an FPGA (Field Programmable Gate Array), instead of being realized by a program.
- ASIC Application Specific Integrated Circuit
- FPGA Field Programmable Gate Array
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Sustainable Development (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Analytical Chemistry (AREA)
- Computer Hardware Design (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Thermal Sciences (AREA)
- Medicinal Chemistry (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
- 1, 110, 120, 130: liquid feed device
- 2: first liquid bottle
- 3: gas pressure adjusting pipeline
- 4, 11, 14, 22, 36, 50, 62, 70, 73, 94: filter
- 5, 7, 18, 37, 44, 57, 63, 67: supply pipe
- 6: pump
- 8: receptacle
- 9, 16, 20, 39, 46, 56, 65, 66: branch point
- 10: first gas introducing valve
- 12: second liquid bottle
- 13: gas pipeline
- 15: second gas introducing valve
- 17, 122: first supply valve
- 19: second supply valve
- 21: liquid supply pipe
- 23, 103: controller
- 31: automatic cell culture device
- 32: thermostat
- 33: refrigerator
- 34: first medium bottle
- 35, 71, 72: gas pressure adjusting pipe
- 38: first control valve
- 42: common pipe
- 43: second medium bottle
- 45: second control valve
- 76: first gas pressure adjusting valve
- 47: gas common pipe
- 40: first gas introducing valve
- 48: humidifying bottle
- 49: second gas pressure adjusting valve
- 51: pressure control valve
- 52: mixed gas cylinder
- 53: first connection
- 54: second connection
- 55: second gas introducing valve
- 58: third gas introducing valve
- 59: gas feed pipe
- 60: gas feed valve
- 61: cell bottle
- 64: third control valve
- 68: fourth control valve
- 69: replenishment bottle
- 74: gas bag
- 75, 131, 132: check valve
- 78, 92, 100: multibranched portion
- 80: first culture container
- 82, 83: container open/close valve
- 81: second culture container
- 84: body portion
- 85: lid portion
- 86: cell suspension
- 87: container
- 88: liquid feed port
- 89: discharge port
- 90: gas pressure adjusting port
- 91: fourth gas pressure adjusting valve
- 92, 100: multibranched portion
- 93: trap bottle
- 96: liquid discharge pump
- 97: liquid discharge bottle
- 98: liquid discharge pipe
- 99: discharge valve
- 101: first container discharge valve
- 102: first container discharge valve
- 111: first weight sensor
- 112, 114, 115: fixing jig
- 113: second weight sensor
- 121: retention pipe
Claims (11)
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2016166681A JP6890389B2 (en) | 2016-08-29 | 2016-08-29 | Liquid transfer device and cell culture device using it |
JP2016-166681 | 2016-08-29 | ||
JPJP2016-166681 | 2016-08-29 | ||
PCT/JP2017/007098 WO2018042710A1 (en) | 2016-08-29 | 2017-02-24 | Liquid feed device, and cell culture device and method using same |
Publications (2)
Publication Number | Publication Date |
---|---|
US20190300837A1 US20190300837A1 (en) | 2019-10-03 |
US11441114B2 true US11441114B2 (en) | 2022-09-13 |
Family
ID=61301779
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/316,908 Active 2038-06-08 US11441114B2 (en) | 2016-08-29 | 2017-02-24 | Liquid feed device, and cell culture device and method using same |
Country Status (4)
Country | Link |
---|---|
US (1) | US11441114B2 (en) |
JP (1) | JP6890389B2 (en) |
CN (1) | CN109563458A (en) |
WO (1) | WO2018042710A1 (en) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP6913650B2 (en) * | 2018-03-14 | 2021-08-04 | 株式会社日立製作所 | Cell culture device |
JP7428866B2 (en) * | 2019-06-20 | 2024-02-07 | シンフォニアテクノロジー株式会社 | Cell collection method and cell culture device |
JP7339138B2 (en) * | 2019-12-02 | 2023-09-05 | 株式会社日立製作所 | Cell culture device |
CN113025481B (en) * | 2021-03-30 | 2023-07-04 | 艾力特生物科技(上海)有限公司 | Automatic fluid infusion device, single fluid infusion method and biological reaction system |
Citations (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4889812A (en) * | 1986-05-12 | 1989-12-26 | C. D. Medical, Inc. | Bioreactor apparatus |
US20060115889A1 (en) | 2004-11-29 | 2006-06-01 | Kawasaki Jukogyo Kabushiki Kaisha | Automatic cell cultivation apparatus having a multijoint robot |
JP2007222120A (en) | 2006-02-27 | 2007-09-06 | Hitachi Medical Corp | Automatic culturing apparatus |
JP2008253154A (en) | 2007-03-30 | 2008-10-23 | Mitsui Eng & Shipbuild Co Ltd | Method for producing alcohol |
US20100317102A1 (en) * | 2006-01-17 | 2010-12-16 | Tsutomu Suzuki | Cell Culture Method and Automatic Culture System Using the Method |
WO2013114845A1 (en) | 2012-02-01 | 2013-08-08 | 東洋製罐グループホールディングス株式会社 | Cell culture kit, and method of using cell culture kit |
WO2015025425A1 (en) | 2013-08-23 | 2015-02-26 | 株式会社日立製作所 | Liquid delivery device and cell culture device using same |
JP2015123012A (en) | 2013-12-26 | 2015-07-06 | 高砂電気工業株式会社 | Micro fluid chip device |
US20150247114A1 (en) * | 2012-09-27 | 2015-09-03 | Ge Healthcare Bio-Sciences Ab | Tangential Flow Perfusion System |
JP2015188381A (en) | 2014-03-28 | 2015-11-02 | 東レエンジニアリング株式会社 | Separation device and separation method |
WO2016013070A1 (en) | 2014-07-23 | 2016-01-28 | 株式会社日立製作所 | Liquid feeding device and cell culture device |
JP2016059316A (en) | 2014-09-17 | 2016-04-25 | 東洋製罐グループホールディングス株式会社 | Liquid feeding method in cell culture system, and cell culture system |
US20170306279A1 (en) * | 2015-01-20 | 2017-10-26 | Fujifilm Corporation | Cell culture device and cell culture method |
-
2016
- 2016-08-29 JP JP2016166681A patent/JP6890389B2/en active Active
-
2017
- 2017-02-24 WO PCT/JP2017/007098 patent/WO2018042710A1/en active Application Filing
- 2017-02-24 US US16/316,908 patent/US11441114B2/en active Active
- 2017-02-24 CN CN201780046793.2A patent/CN109563458A/en active Pending
Patent Citations (18)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4889812A (en) * | 1986-05-12 | 1989-12-26 | C. D. Medical, Inc. | Bioreactor apparatus |
US20060115889A1 (en) | 2004-11-29 | 2006-06-01 | Kawasaki Jukogyo Kabushiki Kaisha | Automatic cell cultivation apparatus having a multijoint robot |
JP2006149268A (en) | 2004-11-29 | 2006-06-15 | Kawasaki Heavy Ind Ltd | Automatic cell-culturing apparatus equipped with multiple joint type robot |
US20100317102A1 (en) * | 2006-01-17 | 2010-12-16 | Tsutomu Suzuki | Cell Culture Method and Automatic Culture System Using the Method |
JP2007222120A (en) | 2006-02-27 | 2007-09-06 | Hitachi Medical Corp | Automatic culturing apparatus |
JP2008253154A (en) | 2007-03-30 | 2008-10-23 | Mitsui Eng & Shipbuild Co Ltd | Method for producing alcohol |
WO2013114845A1 (en) | 2012-02-01 | 2013-08-08 | 東洋製罐グループホールディングス株式会社 | Cell culture kit, and method of using cell culture kit |
US20140335608A1 (en) | 2012-02-01 | 2014-11-13 | Toyo Seikan Group Holdings, Ltd. | Cell culture kit, and method of using cell culture kit |
US20150247114A1 (en) * | 2012-09-27 | 2015-09-03 | Ge Healthcare Bio-Sciences Ab | Tangential Flow Perfusion System |
WO2015025425A1 (en) | 2013-08-23 | 2015-02-26 | 株式会社日立製作所 | Liquid delivery device and cell culture device using same |
US20160108350A1 (en) * | 2013-08-23 | 2016-04-21 | Hitachi, Ltd. | Liquid delivery device and cell culture device using same |
JP2015123012A (en) | 2013-12-26 | 2015-07-06 | 高砂電気工業株式会社 | Micro fluid chip device |
JP2015188381A (en) | 2014-03-28 | 2015-11-02 | 東レエンジニアリング株式会社 | Separation device and separation method |
WO2016013070A1 (en) | 2014-07-23 | 2016-01-28 | 株式会社日立製作所 | Liquid feeding device and cell culture device |
US20170198249A1 (en) | 2014-07-23 | 2017-07-13 | Hitachi, Ltd. | Liquid feeding device and cell culture device |
JP2016059316A (en) | 2014-09-17 | 2016-04-25 | 東洋製罐グループホールディングス株式会社 | Liquid feeding method in cell culture system, and cell culture system |
US20170253847A1 (en) | 2014-09-17 | 2017-09-07 | Toyo Seikan Group Holdings, Ltd. | Liquid delivery method for cell culture system, and cell culture system |
US20170306279A1 (en) * | 2015-01-20 | 2017-10-26 | Fujifilm Corporation | Cell culture device and cell culture method |
Non-Patent Citations (2)
Title |
---|
International Search Report (PCT/ISA/210) issued in PCT Application No. PCT/JP2017/007098 dated May 30, 2017 with English translation (five (5) pages). |
Japanese-language Written Opinion (PCT/ISA/237) issued in PCT Application No. PCT/JP2017/007098 dated May 30, 2017 (six (6) pages). |
Also Published As
Publication number | Publication date |
---|---|
JP6890389B2 (en) | 2021-06-18 |
WO2018042710A1 (en) | 2018-03-08 |
CN109563458A (en) | 2019-04-02 |
JP2018033322A (en) | 2018-03-08 |
US20190300837A1 (en) | 2019-10-03 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11441114B2 (en) | Liquid feed device, and cell culture device and method using same | |
US8281672B2 (en) | Automatable aseptic sample withdrawal system | |
JP7110449B2 (en) | Automated bioreactor systems, systems for automatically performing protocols for decellularization of organs, and waste decontamination systems | |
JP5894260B2 (en) | Culture container and automatic culture device | |
JP2019134725A (en) | Systems and methods for cell culture device interconnection and fluidic device interconnection | |
CA2553332C (en) | Device and method for taking samples | |
JP6062054B2 (en) | Liquid feeding device and cell culture device using the same | |
EP3260527A1 (en) | Cell culture device, cartridge for culture medium replacement use, and method for replacing culture medium | |
RU2014137564A (en) | AUTOMATIC AND AUTOMATED METHOD OF CULTIVATION OF CELLS | |
CN107739713A (en) | It is a kind of to change liquid system suitable for what spatial cell was cultivated automatically | |
JP5960256B2 (en) | Culture container and automatic culture device | |
CN207525252U (en) | Liquid system is changed suitable for what spatial cell was cultivated automatically | |
JP6514952B2 (en) | Automatic culture device | |
JP6294485B2 (en) | Liquid feeding device and cell culture device | |
US10947490B2 (en) | Liquid delivery device and cell culture device using the same | |
US11814614B2 (en) | Cell culture device | |
US20210130763A1 (en) | Device and method for sterile sample-taking | |
CN213266493U (en) | Sampling device | |
US20220411739A1 (en) | Genetically modified cell production system | |
WO2018105078A1 (en) | Cell culture method, culture vessel, and cell culture apparatus | |
JP2004208664A (en) | Cell culture system, and method for diluting/sampling cultured cell |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: HITACHI, LTD., JAPAN Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:KIYAMA, MASAHARU;ZHOU, GUANGBIN;SUZUKI, DAISUKE;AND OTHERS;SIGNING DATES FROM 20181015 TO 20181016;REEL/FRAME:047959/0539 |
|
FEPP | Fee payment procedure |
Free format text: ENTITY STATUS SET TO UNDISCOUNTED (ORIGINAL EVENT CODE: BIG.); ENTITY STATUS OF PATENT OWNER: LARGE ENTITY |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: FINAL REJECTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: ADVISORY ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NOTICE OF ALLOWANCE MAILED -- APPLICATION RECEIVED IN OFFICE OF PUBLICATIONS |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: PUBLICATIONS -- ISSUE FEE PAYMENT VERIFIED |
|
STCF | Information on status: patent grant |
Free format text: PATENTED CASE |